Enhancing tetracycline antibiotic blood levels with pyrazolidine potentiating agentstherefor



United States Patent Office 3,296,073 Patented Jan. 3, 1967 Delaware No Drawing. Filed Feb. 5, 1964, Ser. No. 342,783 17 Claims. (Cl. 167-65) This invention relates to novel antibiotic compositions which comprise a tetracycline antibiotic together with a heterocyclic potentiating agent therefor. More particularly, it is concerned with oral compositions of the foregoing type which all contain a pyrazolidine-type compound for the aforestated purpose, whereby there is thus achieved an enhancement of said antibiotic blood levels in the animal to whom they are so administered.

In this connection, it is to be understood that by the use of the term a tetracycline antibiotic there is meant to be included not only such amphoteric tetracycline compounds as chlortetracycline, tetracycline, oxytetracycline, 6-demethylchlortetracycline, 6-deoxytetracycline, 6-demethyl- 6 deoxytetracyc-line, 6-demethyl-6-deoxy-7-bromtetracycline, 6-demethyl-6-deoxy-6-methylene-5-oxytetracycline, etc., but also such acid addition salts thereof as the hydrochloride, hydrobromide, hydriodide, sulfate, nitrate, phosphate, acetate, lactate, maleate, citrate, tartrate and ascorbate, as well as base salts of same such as the sodium, potassium, calcium, quaternary ammonium or ethylene diamine salts thereof, or the aluminum gluconate complex or other organic acid complex salts thereof, or any other pharmaceutically acceptable salts of these particular antibiotics which happen to be therapeutically useful per se.

The pyrazolidine-type compounds of these novel compositions are all l,2-diphenyl-4-( l'-cyano)cycloalkylpyrazolidine-3,5-diones which are reported to be useful as antiinflammatory agents. These compounds together with other related types are presently being claimed in copending U.S. patent application Serial No. 135,421, filed September l, 1961 by D. P. Cameron, now U.S. Patent No. 3,133,934. Key compounds in the present instance for use in this invention are 1,2-diphenyl-4-(l-cyano)-cyclopentylpyrazolidine-3,S-dione, 1,2-diphenyl-4-(1'-cyano)cyclohexylpyrazolidine-3 ,5 -dione, l,2-diphenyl-4-( l -cyano-4'- methyl)cyclohexylpyrazo-lidine-3,S-dione and 1,2-diphenyl-4-(1-cyano)cycloheptylpyrazolidine-3,5-dione, including their pharmaceutically acceptable base salts as well, such as the alkali metal and alkaline-earth metal salts thereof.

In general, the amount of pyrazolidine-type compound to be used as potentiating agent for the tetracycline component of the mixture may vary over a wide range, but it is usually more desirable in practice to employ at least a semiequal amount by weight of the pyrazolidine-type compound and preferably, even an equal amount or excess of same as it is the cheaper of the two components. Optimum results, however, have been obtained with from about one-half to about ten parts by weight of the pyrazolidine-type compound with respect to about one part by weight of the tetracycline component. As previously indicated, all these compositions give remarkably highly enhanced antibiotic blood levels so that it is now possible, i.e., within the realm of reality, to achieve much higher tetracycline antibiotic concentrations in the blood of the animals to whom they are so administered with a given amount of antibiotic compound than was heretofore ever thought possible or conversely, to achieve the same levels The advantages afforded the use of this invention are therefore manifestly obvious. Moreover, the pyrazolidine potentiating agent can be administered either before or after the administration of the antibiotic provided the time interval between the two is not too excessive, i.e., not more than about 1020 minutes. For instance, incorporation of 1,2-diphenyl-4-( l-cyano)c-yclohexylpyrazolidine-3,5-dione into the feed alone without the added antibiotic, followed by the intramuscular injection of oxytetracycline hydrochloride into the so fed animal results in a potentiating effect with respect to the tetracycline antibiotic blood levels. However, it is preferred in practice to use the two main components of the mixture together, i.e., at the same time, as will hereinafter be discussed in more detail.

A preferred unit dosage form for administering these novel compositions is most conveniently a powdered mixture of the two principal components encased in a soft or hard-shelled gelatin capsule. This dosage form may contain from about 25 mg. up to about 500 mg. of the desired tetracycline together with from about one-half to about ten parts by weight of the chosen pyrazolidine-type compound. An inert diluent, such as a pharmaceuticallyacceptable inert carrier like starch, lactose or milk sugar, or glucosamine, may also be present if so desired. Conversely, it is also possible to administer these compositions in a granulated form as such or else compressed into tablets for oral administration, as well as in the form of aqueous suspensions, elixirs, lozenges, troches, hard candies and pediatric drops, etc. When administered parenterally, they can be given to the animal in the form of an aqueous dispersion or solution for intravenous injection, or as an organic solution or suspension for intramuscular injection.

Moreover, these compositions may also be administered to domesticated animals, such as rats, dogs, sheep, chickens, turkeys, ducks, hogs, rabbits, and the like by way of their feeds or drinking water in addition to the direct route previously indicated. For instance, the oral unit dosage forms previously discussed may be added as such to the animal feeds or, and preferably, the novel compositions themselves may be blended into powders for incorporation directly into the feeds. The antibiotic component of the feed may vary anywhere from about 50 g. up to about 1000 g. per ton of feed, While the pyrazolidine potentiating agent must be present in the mixture to the extent that it is at least about 0.1% by weight based on the total weight of the composition. Needless to say, the tetracycline-type antibiotic component and the pyrazolidine potentiating agent therefor may either be administered via one component of the animals feed or else they may be uniformly blended together or distributed therein throughout a completely-mixed feed. In this latter connection, it is to be noted that a wide variety of different feed components may be of use in the nutritional diets called for. Thus, the proper feed compositions should normally contain nutritionally-balanced amounts of carbohydrate, fat and protein, together with the usual vitaminaceous and mineral supplementary sources.

When the novel compositions of this invention are to be used in animal feeds, a particular type of conventional feed material for just such purposes is one which generally follows the suggested formulae set forth on page six of the authoritative pamphlet issued by the National Research Council at Washington, DC, June 1944, and entitled Recommended Nutrient Allowances for Poultry.' For instance, feed compositions are recommended to contain roughly from between about 50% and about of grains, such as ground grain or grain by-products, molasses and other sugar by-products; from between about 3% and about 10% of animal protein, such as fish meal, meatscraps, and the like; from between about and about 30% ofvegetable protein, such as soybean oil meal, peanut meal, cottonseed meal, and the like; and from between about 2% and about 5% of minerals, such as bone meal, limestone, and so forth; together with supplementary vitaminaceous sources, such as thiamine, riboflavin, niacin, etc. Furthermore, the compositions of this invention may either be used alone in said feeds or else in conjunction with various other antibiotics and chemotherapeutic agents, such as penicillin, bacitracin, sulfaquinoxaline, phenylarsonic acid, hydroxyzine, oleandornycin, and so forth.

In accordance with a more specific embodiment of this invention, a typical feedstuff for poultry may be prepared by mixing the following ingredients together in the proportions by weight specified below:

The trace mineral mix constitutes 0.05% by weight of the feed and the ingredients contained therein are as follows together with their weight percentages in the premix: manganese, 24% iodine, 0.48% iron, 8.0% copper, 0.8% zine, 0.04% and cobalt, 0.08%.

The vitamin premix constitutes 0.55% by weight of the feed and the ingredients contained therein are listed below as follows together with their percent concentrations in the premix:

Percent Vitamin A (10,000 I.U./g.) 10.0 Vitamin D3 (3,000 I.C.U./g.) 10.0 Choline chloride (25%) 35.25 Niacin (80%) 0.5 DL-Calcium pantOthena-te (45%) 10.0 Riboflavin (4 g./lb.) 0.39 Vitamin B12 (60 ing/lb.) 0.20 Vigofac* 30.0 Fine corn meal 3.75

*Vigofac is the registered trademark name of the 'Chas. Pfizer & 00., Inc. for unidentified poultry-livestock growth factors obtained from Streptomyces fermentation sources.

Needless to say, many other comparable feeds may also be blended together in a similar fashion, such as, for example, those which contain lesser amounts of calcium, for instance, up to about 0.5% by weight of calcium as compared to the 1.2% calcium concentration level which is contained in the feeds of the foregoing description.

The invention is further illustrated by the following examples, which are not to be construed in any way or manner as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications and equivalents thereof which readily suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

EXAMPLE I Ten eight-week old chickens (five male and five female) of the Vantress-White Rock Cross broiler variety were each fed a standard animal feedstuff diet of the type described previously, which also contained 400 g. of oxytetracycline hydrochloride per ton, together with 1,2-disamples to determine the oxytetracycline blood sera levels therein. The results obtained in this manner are shown below in the following table:

Average Oxytetra- Potentiating Efieet cycline Blood Sera (Index) Substance Levels (meg/m1.)

24 hrs. 72 hrs. 24 hrs. 72 hrs.

Oxytetracyeline HCl 0. 2280 0. 2140 100 100 Oxytetracycline HCl plus Compound N o. 1 0. 2535 0.2832 114 132 From the data presented in the above table, it is obvious that l,2-diphenyl-4-( 1-cyano) cyclohexylpyrazolidine-3,5- dione (Compound No. 1) produced an increase in oxytetracycline blood sera levels over that of the controls during both assay periods.

EXAMPLE II Fifty (50) six-week old chickens of the Vantress-White Rock Cross broiler variety were divided into ten groups of five male and five female chicks each and subjected to the general procedure of Example I except for the fact that different amounts of l,2-diphenyl-4-(l'-cyano)cyclohexylpyrazolidine-3,5-dione (Compound No. 1) were employed. The results obtained in this manner are summarized below in the following table:

The procedure described in the preceding example is essentially followed except that sixty (60) seven-week old chickens of the Vantress-White Rock Cross broiler variety are used this time instead and the weight concentrations of the added pyrazolidine-type compound are somewhat different. One-half of the chickens in each treatment group were male and one-half female, with a total of twenty chickens being used in the control group. The results obtained in this manner are all summarized below in the following table:

Average Oxytetra- Potentiating Efiect Conan. eyeline Blood Sara (Index) Potentiating Level, Levels (meg/ml.)

Compound Percent 24 hrs. 72 hrs. 24 hrs 72 hrs.

None 0. 169 0. 193 100 Cpd. No. 1 0. 267 0. 287 158 148 Cpd. N 0. 0. 321 0. 368 191 Cpd. No. 1- 0.276 0. 448 163 231 Cpd. N o. 1 1. 000 0. 299 0. 440 177 228 From the data presented in this table, it is obvious that 1,2 diphenyl 4 (1 cyano) cyclohexylpyrazolidine-3,5- dione (Cpd. No. 1) potentiates oxytetracycline at levels as low as 0.125% by weight.

EXAMPLE IV Twenty (20) seven-week-old chickens of the type employed in the previous example were divided equally by sex into two groups of ten chickens each. One group was maintained on the control ration which was the same standard feed ration used before in Examples I-III. The other group was fed the control ration initially, but later was switched to a feed ration of the same type containing 1,2-diphenyl 4 (1-eyano)cyclohexylpyrazolidine- "and six hours, the following results were obtained in accordance with the procedure of Example I:

Average Oxytetr Potentiation Efiect cycline Blood Sera (Index) Compound in Feed Levels (meg/m1.)

2 hrs. 6 hrs. I 2 hrs. 6 hrs.

None 0. 369 0. 108 100 100 Cpd. No. 1 0. 481 0.217 130 201 From the above table, it is obvious that 1,2-diphenyl- 4-(l'-cyano)cyclohexylpyrazolidine-3,5dione (Cpd. No. l) in the feed potentiates oxytetracycline blood sera levels when the latter antibiotic compound is given intramuscularly.

EXAMPLE V The procedure described in Example I was repeated except that 1,2-diphenyl 4 (1'-cyano)cyclohexylpyrazolidine-3,5-dione (Compound No. 1) was employed at the 0.3% concentration level and tests with chlortetracycline hydrochloride in the feed (instead of oxytetracycline hydrochloride) were included for comparison purposes. The animals employed, totalling forty (40) in number, were all five-week-old broiler strain chicks of the Vantress-White Rock Cross variety. They were divided into four groups of ten animals each (five male and five female). The first two groups received the oxytetracycline-containing feed with and without added Compound No. 1, while the other two groups received the chlortetracycline-containing feed, also at 400 grams/ ton, and with and without the aforementioned pyrazolidine-type compound (Cpd. No. 1). At the end of 24 and 72 hours, the birds were bled by heart puncture and the following results were obtained:

From the above table, it can be seen that chlortetracycline hydrochloride is potentiated in feeds by 1,2-diphenyl-4-( 1'-cyano)cyclohexylpyrazolidine 3,5 dione (Compound No. 1) to an even greater extent than is oxytetracycline hydrochloride.

EXAMPLE VI Twenty-one (21) eleven-week-old female Vantress- White Rock Cross broilers are divided into three pens of seven birds each. These birds are fed the same standard feed diet as described before and given Water continuously throughout the entire test period. One group received an oral dose of oxytetracycline hydrochloride at the 250 mg. level together with one-half that amount by weight of 1,2-diphenyl 4 (l'-cyauo)cyclohexylpyrazolidine-3,5- dione (Compound No. 1), while another group received the same two chemicals in a 1:2 weight ratio, respectively, i.e., 250 mg. of oxytetracycline hydrochloride and 500 mg. of Compound No. 1. The remaining group of animals served as the controls and are given the antibiotic only without the added pyrazolidine-type compound. At the end of two and siX hours after the initial oral dosing, oxytetracycline blood serum levels are determined by standard assay procedures and the results obtained indicate that 1,2-diphenyl-4-(1'-cyano)cyclohexylpyrazolidine-3,5-dione gives significantly greater blood levels with oxytetracycline than does the antibiotic alone.

EXAMPLE VII The procedure described in Example VI is followed except that 1,2-diphrenyl 4 r (1 cyano)cyclopentylpyrazolidine-3,5-dione is employed in place of the pyrazolidine-type compound (Cpd. No. 1) previously used. In like manner, 1,2-diphenyl-4-(l'-cyano)cycloheptylpyrazolidine-3,5-dione and 1,2-dipheny1-4-(l-cyano-4'-methyl) cyclohexylpyrazolidine-3,S-dione are also individually employed. with all these compounds being used on the same weight basis as Cpd. No. 1. In each and every case, the results obtained indicate that these closely-related homologs of Cpd. No. 1 all act in substantially the same manner, i.e., they afford enhanced antibiotic blood levels.

EXAMPLE VIII The procedure described in Example V1 is followed here except that chlortetracycline is now employed in place of oxytetracycline on the same weight basis as the antibiotic of choice. In like manner, tetracycline hydrochloride is also used. In both these instances, comparable results with respect to oxytetracycline are obtained.

EXAMPLE IX The procedure described in Example VI is repeated with forty-two (42) twelve-week-old female Vantress- White Rock broilers divided into groups of six birds each. In this case, the oxytetracycline hydrochloride is employed at the 25 mg. oral dose level and the 1,2-diphenyl- 4-(l-cyano)cyclohexylpyrazolidine 3,5 dione is employed at various levels ranging from mg. to 320 mg. in either 5 ml. or 10 ml. amounts (of water). The results obtained in this manner indicate that at the end of two and six hours after administration, 1,2-diphenyl-4- (1'-cyano)cyclohexylpyzraz'olidrine-3,5-dione is an eifective potentiating agent for oxytetracycline by the oral route.

EXAMPLE X The procedure described in the preceding example is followed except that 500 mg. of oxytetracycline hydrochloride is employed instead of 25 mg. In like manner, tetracycline hydrochloride and chlortetracycline hydrochloride are each similarly employed at both these same dose levels, i.e., at 25 mg. and 500 mg. respectively. In all these cases, the results obtained are substantially the same with respect to degree of potentiation as those previously obtained in Example IX. Comparable results are also obtained in all these cases when 1,2-diphenyl-4- (1 cyano)cyclopentylpyrazolidine 3,5 dione, 1,2-diphenyl-4-(1-cyano 4' methyl)cyclohexylpyrazolidine 3,5-dione and 1,2-diphenyl-4- 1'-cyano) cycloheptylpyrazolidine-3,5-dione are all each individually employed in place of the corresponding cyclohexyl compound.

EXAMPLE XI Nine fasting dogs, each weighing anywhere from about 15 to about 49 pounds are used in this experiment. The animals each received an experimental capsule containing 250 mg. of oxytetracycline hydrochloride together with 250 mg. of 1,2-diphenyl-4-(1-cyano)cyclohexylpyrazolidine-3,5-dione. At the end of 3 and 6 hours after capsule administration, blood samples for serum assay are obtained from the dogs and analyzed in the usual fashion. The results obtained in this manner indicate that 1,2-diphenyl-4-(1'-cyano)cyclohexylpyrazolidine 3,5-dione is definitely effective as a potentiator of oxytetracycline blood levels in said animals.

7 EXAMPLE XII Forty-eight (48) eight-week old chickens of the Vantress-White Rock Cross broiler variety are divided into two groups of four male and four female birds each. They are then fed the standard basal animal feedstuff diet without added antibiotic and subject to the usual treatment as described before, employing oxytetracycline hydrochloride at the 25 mg. dose level and 1,2-diphenyl-4- (1'-cyano)cyclohexylpyrazolidine-3,5-dione at the same dose level as well as at 500 mg., and including both water solution and capsule form for the afore-mentioned pyrazolidine-type compound. The results obtained in this manner at the end of 3 and 6 hours after initial drug administration indicate that oxytetracycline potentiating effects are obtained with 250 mg. and 500 mg. capsule doses of the pyrazolidine-type compound, respectively, and that the results obtained in the case of the aqueous solutions are only slightly less. Further, tests performed with the closely-related homologs of this potentiating heterocyclic compound, viz., 1,2-diphenyl-4-(1-cyano)cyclopentylpyrazolidine 3,5 dione, 1,2-diphenyl-4-(1'-cyano-4-methyl)cyclohexylpyrazolidine-3,S-dione and 1,2-diphenyl-4- (1-cyano)cycloheptylpyrazolidine 3,5 dione, also indicate market potentiating effects for these particular compounds.

EXAMPLE XIII Sixty (60) eight-week old chickens of the Vantress- White Rock Cross broiler variety are divided into six groups of five male and five female birds each and fed the standard diet ration used before. One set of three such groups received oxytetracycline hydrochloride at levels of 200, 500 and 1000 grams per ton, respectively, together 1,2 diphenyl 4 (1-cyano)cyclohexylpyrazolidine-3, S-dione at the 2.0% concentration level. The other set of birds received the antibiotic alone, without the added pyrazolidine-type compound, at the same three antibiotic dose levels previously indicated. At the end of 24, 48 and 72 hours after initiation of the feeding, the birds are bled by heart puncture in order to run blood sera assays on the resulting samples. The results obtained in this manner indicate that 1,2-diphenyl-4-(1'-cyano)cyclohexylpyrazolidine-3,5-dione at the 2.0% concentration level potentiates oxytetracycline at all three antibiotic. dose levels tested.

EXAMPLE XIV The procedure described in Example XIII is repeated except that oxytetracycline hydrochloride is now employed at concentration levels of 50, 100 and 150 grams per ton, respectively. In each and every case, the results obtained are comparable in order of magnitude to those of the foregoing example achieved with the higher concentration values.

EXAMPLE XV Forty-eight (48) young chickens of the Vantress-White Rock broiler variety are placed into pens of six male and six female birds each. The animals are then fed the standard animal basal feedstuflf ration, with one group of 12 animals receiving oxytetracycline at the 400 g. per ton level together with l,2-dip'henyl-4-(1-cyano)cyclohexylpyrazolidine-3,5-dione at the 2% concentration level, while another group of twelve served as the controls and received no such added compound. In like manner, these tests are again repeated with two other groups of twelve animals only this time employing chlortetracycline in place of the oxytetracycline. At the end of 24 and 72 hours after initiation of said treatment, the birds are bled in order to determine blood sera levels on their samples. The results obtained in this manner indicate that 1,2-diphenyl-4-(1-cyano)cyclohexylpyrazolidine-3,S-dione pro duces potentiation of both the oxytetracycline and chlortetracycline blood levels at the end of each of the two aforementioned time periods.

8 EXAMPLE XVI A group of five albino rats received an initial dose of 6-demethyl-6-deoxy-6-methylene-5-oxytetracycline hydrochloride orally via an oral tube at the 125 mg./kg. level. A second group received the same dose together with an equal amount by weight of 1,2-diphenyl-4-(1-cyano)- cyclohexylpyrazoline-3,S-dione (125 mg./kg.). A third group received no antibiotic nor added compound what soever and served as the controls, while a fourth group received the heterocyclic compound alone. The animals are thereafter placed in metabolism cages for 18 hours post close and the total volume of urine excreted is collected. Food is then withheld from the animals throughout this entire test period, but water is given them ad libitum. The results obtained in this manner indicate that 1,2-diphenyl 4 (1-cyano)cyclohexylpyrazolidine-3,5- dione at the 125 mg./kg. dose level gives much greater urinary excretion levels with 6-demethyl-6-deoxy-6-methylene-5-oxytetracycline hydrochloride than does the antibiotic itself when used alone.

EXAMPLE XVII The procedure described in Example IV is repeated using other pyrazolidine dione compounds in place of 1,2- diphenyl-4-(l'-cyano)cyclohexylpyrazolidine 3,5 dione on an individual basis. These other compounds specifically are 1,2 diphenyl-4-(1-cyano)cyclopentylpyrazolidine-3,5-dione, 1,2-diphenyl-4-( 1-cyano-4'-methyl)cyclohexylpyrazolidine 3,5 dione and 1,2-diphenyl -4 (1- cyano)cycloheptylpyrazolidine 3,5 dione. In each and every case, the results obtained show a marked enhancement of oxytetracycline antibiotic blood levels.

In like manner, the substitution of both chlortetracycline as well as tetracycline for oxytetracycline in this procedure gives equally similar results.

EXAMPLE XVIII Seventy (70) Broiler-type chicks were given continuous access to the normal feed diet (with no antibiotic added) and water. Each bird was also dosed with 10 ml. of a propylene glycol suspension containing mg. of oxytetracycline hydrochloride, except that in the case of fifty of these birds (the birds were divided into groups of ten) pyrazolidine dione potentiating agents were blended into the oxytetracycline-propylene glycol dosage form at levels of either 100 mg. or 250 mg. as the case may be. The other twentybirds, of course, served as the controls. The pyrazolidine dione compounds employed here were 1,2-

diphenyl-4-(1-cyano)cyclopentylpyrazoline 3,5 dione (Compound No. 2), 1,2-diphenyl-4-(1'-cyano)cycloheptylpyrazolidine-3,5-dione (Compound No. 3) and 1,2-diphenyl-4-(1'-cyano 4' methyl)cyclohexylpyrazolidine- 3,5-dione (Compound 'No. 4), and the results obtained are listed below as follows where the potentiation effect is reported at the end of six and 24 hours after initial dosing, based on oxytetracycline blood sera levels determined in the usual fashion:

1. An antibiotic composition comprising a tetracycline antibiotic and a compound selected from the group consisting of 1,2 diphenyl-4-(1-cyano)cyclopentylpyrazolidine-3,5-dione, 1,2 diphenyl-4-(l-cyano)cyclohexylpyrazolidine-3,5-dione, 1,2 diphenyl-4-(1'-cyano-4-methyl)cycloheXylpyrazlidine-3,S-dione and 1,2 diphenyl-4- l'-cyano) cycloheptylpyrazolidine-3 ,5 -dione.

2. A composition as claimed in claim 1 wherein the antibiotic is oxytetracycline.

3. A composition as claimed in claim 1 wherein the antibiotic is tetracycline.

4. A composition as claimed in claim 1 wherein the antibiotic is chlortetracycline.

5. An antibiotic composition as claimed in claim 1 wherein the amount of pyrazolidine compound employed is at least equal to one-half that of the tetracycline antibiotic.

6. An antibiotic composition in unit dosage form suitable for oral administration, comprising from about 25 mg. up to about 500 mg. of a tetracycline antibiotic and from about one-half to about ten parts by weight of a compound selected from the group consisting of 1,2-diphenyl-4-(1'-cyano)cyclopentylpyrazolidine-3,S-dione, 1, 2 diphenyl 4 (1-cyano)cyclohexylpyrazolidine-3,5- dione, 1,2 diphenyl-4-(1'-cyan0-4'-methyl)cyclohexylpyrazolidine-3,5-dione and 1,2-diphenyl-4-(1'-cyano)cyc1oheptylpyrazolidine-3,5-dione with respect to the tetracycline antibiotic.

7. A composition as claimed in claim 6 wherein the pyrazolidine compound is 1,2-diphenyl-4-(lcyano)cyclopentylpyrazolidine-3,S-dione.

8. A composition as claimed in claim 6 wherein the pyrazolidine compound is 1,2-diphenyl-4-(1-cyano)cyclohexylpyrazolidine-3,S-dione.

9. A composition as claimed in claim 6 wherein the pyrazolidine compound is 1,2-diphenyl 4 (1-cyano-4- methyl)cyclohexylpyrazolidine-B,S-dione.

10. A composition as claimed in claim 6 wherein the pyrazolidine compound is 1,2-diphenyl-4-(1-cya11o)cycloheptylpyrazolidine-3,S-dione.

11. An animal feed composition comprising a nutritionally-balanced animal feed to which there is added a tetracycline antibiotic and a compound selected from the group consisting of 1,2-diphenyl-4-(1'-cyano)cyclopenylpyrazolidine-3,5-dione, 1,2 diphenyl-4-(1'-cyano)cyclohexylpyrazolidine-3,S-dione, 1,2 diphenyl-4-(1'-cyano-4'- methyl)cycloheXylpyraZolidine-3,S-dione and 1,2-diphenyl-4-(1'-cyano)cycloheptylpyrazolidine-3,S-dione.

12. A feed composition as claimed in claim 11 wherein the antibiotic is present at a concentration level that is in the range of from about g. up to about 1000 g. per ton of said feed.

13. A feed composition as claimed in claim 11 wherein the pyrazolidine compound is present to the extent that it is at least about 0.1% by Weight of the total composition.

14. A method of potentiating the blood levels of a tetracycline antibiotic, which comprises the step of administen'ng the tetracycline antibiotic in conjunction with a compound selected from the group consisting of 1,2-diphenyl-4-(1'-cyano)cyclopentylpyrazo1idine-3,5 dione, l, 2 diphenyl 4 (1'-cyano)cyclohexylpyrazolidine 3,5- dione, 1,2 diphenyl-4-(1-cyano-4-methyl)cyclohexylpyrazo1idine-3,5-dione and 1,2-dipheny1-4-(1'-cyano)cycloheptylpyrazolidine-3,5-dione.

15. A method as claimed in claim 14 wherein the tetracycline antibiotic is orally administered.

16. A method as claimed in claim 14 wherein the tetracycline antibiotic is parenterally administered.

17. A method as claimed in claim 14 wherein the amount of pyrazolidine compound employed is at least equal to one-half by weight of that of the tetracyclinetype antibiotic.

References Cited by the Examiner UNITED STATES PATENTS 3,133,934 5/1964 Cameron 260-310 LEWIS GOTTS, Primary Examiner.

S. K. ROSE, Assistant Examiner. 

1. AN ANTIBIOTIC COMPOSITION COMPRISING A TETRACYCLINE ANTIBIOTIC AND A COMPOUND SELECTED FROM THE GROUP CONSISTING OF 1,2-DIPHENYL-4-(1''-CYANO)CYCLOPENTYLPYRAZOLIDINE-3,5-DIONE, 1,2-DIPHENYL-4-(1''-CYANO)CYCLOHEXYLPYRAZOLIDINE-3,5-DIONE, 1,2-DIPHENYL-4-(1''-CYANO-4''-METHYL)CYCLOHEXYLPYRAZOLIDINE-3,5-DIONE AND 1,2-DIPHENYL-4(1''-CYANO)CYCLOHEPTYLIPYRAZOLIDINE-3,5-DIONE. 